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Objective:To determine fatty acid synthase(FAS)expression in human multiple myeloma and verify its potential as a therapeutic target in multiple myeloma.Methods:FAS expression was determined by immunohistochemistry,re- verse-transcription polymerase chain reaction(RT-PCR)and immunoblot analysis in bone marrow samples obtained from 27 patients with multiple myeloma(MM patients)and peripheral blood mononuclear cells(PBMCs)obtained from 12 healthy donors. In parallel,additional analyses were performed on 2 human multiple myeloma cell lines,U266 and RPMI8226.U266 cells were treated with cerulenin at various concentrations(5 to 320μg/ml)for 24 h,and metabolic activity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assays.Apoptosis was evaluated by dual Annexin V/PI (propidium iodide)labeling and flow cytometry(FCM)in U266 cells treated with 20μg/ml cerulenin for 12 h or 24 h.Results:By immunohistochemistry,we found that 19 of 27 bone marrow samples obtained from MM patients expressed significantly high levels of FAS.Similarly,by RT-PCR,22 of 27 bone marrow samples obtained from MM patients,U266 and RPMI8226 showed FAS expression,whereas PBMC samples from 12 healthy donors did not express detectable level of FAS.FAS protein expression was confirmed by immunoblot analysis in 16 of 27 bone marrow samples obtained from MM patients,U266 and RPMI8226 cell lines,and no FAS protein expression was detected in PBMC samples from 12 healthy donors.U266 cells were highly sensitive to cerulenin treatment,with a dosage-related effect on metabolic activity,as a measure for cell proliferation.U266 cells treated with 20μg/ml cerulenin for 12 and 24 h also showed early sign of apoptosis with 56.9%and 69.3%Annexin V+/PI ?cells,and late apoptotic and necrotic cells with 3.2%and 17.6%Annexin V+/PI +cells.Conclusion:Increased FAS expression existed in multiple myeloma samples and human myeloma cell lines.Cerulenin greatly inhibited metabolic activity/cell proliferation of U266 cells and induced apoptosis,suggesting that FAS is an effective target for pharmacological therapy in human multiple myeloma.
Objective: To determine fatty acid synthase (FAS) expression in human multiple myeloma and verify its potential as a therapeutic target in multiple myeloma. Methods: FAS expression was determined by immunohistochemistry, re- verse-transcription polymerase chain reaction (RT-PCR) and Immunoblot analysis in bone marrow samples obtained from 27 patients with multiple myeloma (MM patients) and peripheral blood mononuclear cells (PBMCs) from 12 healthy donors. In parallel, additional analyzes were performed on 2 human multiple myeloma cell lines, U266 and RPMI8226. U266 cells were treated with cerulenin at various concentrations (5 to 320 μg / ml) for 24 h, and metabolic activity was measured by 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide . Apoptosis was evaluated by dual Annexin V / PI (propidium iodide) labeling and flow cytometry (FCM) in U266 cells treated with 20 μg / ml cerulenin for 12 h or 24 h. Results: By immunohistochemistry, we found that 19 of 27 bone marrow samples obtain ed from MM patients showed significantly high levels of FAS.Similarly, by RT-PCR, 22 of 27 bone marrow samples obtained from MM patients, U266 and RPMI8226 showed FAS expression, whereas PBMC samples from 12 healthy donors did not express detectable level of FAS .FAS protein expression was confirmed by immunoblot analysis in 16 patients with bone marrow samples obtained from MM patients, U266 and RPMI8226 cell lines, and no FAS protein expression was detected in PBMC samples from 12 healthy donors. U266 cells were highly sensitive to cerulenin treatment , with a dosage-related effect on metabolic activity, as a measure for cell proliferation. U266 cells treated with 20 μg / ml cerulenin for 12 and 24 h also showed early sign of apoptosis with 56.9% and 69.3% Annexin V + / PI? cells, and late apoptotic and necrotic cells with 3.2% and 17.6% Annexin V + / PI + cells. Confluence: Increased FAS expression existed in multiple myeloma samples and human myeloma cell lines. Cerulenin greatly reduced metabolic activity / cell p roliferation of U266 cells and induced apoptosis, suggesting that FAS is an effective target for pharmacological therapy in human multiple myeloma.